1. Technical Field
The present invention is related to a method of quantitation of activation of the human terminal complement cascade. More particularly, the present invention is related to a kit and a quantitative method for fluid phase human terminal complement cascade activation by enzyme-linked immunosorbent assay.
2. State of the Art
It has been reported that the terminal complement cascade is activated in a variety of human diseases, including the glomerulonephritis and cutaneous lesions of systemic lupus erythematosus (Biesecker et al. J. Exp. Med. 154: 1779, 1981; Biesecker et al., N. Engl. J. Med. 306: 264, 1982; Falk et al. Clin. Research 32:503A (Abstract), 1984), other glomerulonephritides (Falk et al. J. Clin. Invest. 72:560, 1983), bullous pemphigoid (Dahl et al. J. Invest. Dermatol. 82:132, 1984), dermatitis herpetiformis (Dahl et al. Arch. Dermatol. 121:70, 1985), and demyelinating diseases (Sanders et al. Clin. Research 33:388A (abstract), 1985). However, a quantitative and sensitive enzyme linked immunosorbent assay to determine or detect the activation of the terminal cascade in the human body fluid is not available. Therefore, the availability of a simple, sensitive assay which measures activation of the terminal complement components should prove useful for studying the pathophysiology of a variety of disease states and for monitoring disease activity.
Activation of the terminal portion of the complement cascade results in the assembly of a macromolecular complex consisting of C5b, C6, C7, C8 and a variable number of C9 monomers. If activation occurs in the fluid phase, two or three molecules of S protein combine with the complex, making it cytolytically inactive (Kolb and Muller-Eberhard Proc. Natl. Acad. Sci. U.S.A. 72:1687, 1975). However, this SC5b-9 complex is water soluble and contains 2-3 C9 monomers. In the presence of a target membrane, the activation of the terminal complement cascade results in the formation of the MC5b-9 complex, which is cytolytically active (Mayer Proc. Natl. Acad. Sci. 69:2954, 1972). The MC5b-9 complex contains a variable number of C9 monomers, as many as 12 to 16, depending on the availability of C9 (Podack et al. J. Exp. Med. 156:268, 1982; Bhakdi and Tranum-Jensen J. Immunol. 133:1453, 1984). Furthermore, purified human C9 alone if incubated at 37.degree. C. spontaneously forms closed-ring, SDS-resistant polymerized C9 (poly C9) (Podack and Tschopp Proc. Natl. Acad. Sci. U.S.A. 79:574, 1982) and polymerization of purified C9 results in expression of a C9 neoantigen. This C9 neoantigen- is also expressed in SC5b-9 and MC5b-9 complexes (Podack and Tschopp J. Biol. Chem. 257:15204, 1982; Falk et al. J. Clin Invest. 72:560, 1983), as are several other neoantigens related to other steps in C5b-9 assembly (Kolb and Muller-Eberhard J. Exp. Med. 141:734, 1975).
Polyclonal antibodies directed to all of the neoantigens of SC5b-9 have been used in an immunoradiometric assay for SC5b-9 (Bhakdi and Muhly J. Immunol. Methods 57:283, 1983). This assay, which is based on the inhibition of binding of radiolabeled antineoantigen antibodies to rabbit erythrocyte membranes bearing MC5b-9 lesions, is sensitive to 3 to 4 .mu.g of SC5b-9 per ml, equivalent to a 1% activation of the terminal complement components in normal serum. In another competitive inhibition radioimmunoassay for SC5b-9 (Falk et al. Clin. Research 32:503A (abstract), 1984), a monoclonal antibody to the C9 neoantigen was used. In this assay, radiolabeled polymerized C9 was displaced from the monoclonal antibody by the SC5b-9 present in the test sample. A standard curve was created using unlabeled poly C9, and the results were expressed as unit equivalents of poly C9 rather than as units of SC5b-9. The sensitivity of the assay was not reported. Finally, an enzyme-linked immunosorbent assay (ELISA) for SC5b-9 has been described which, in a sandwich fashion, uses antibodies to native epitopes in two different complement components present in the assembled C5b-9 complex. Although this approach yields a sensitive assay for SC5b-9, the utility of the assay is limited in that its signal can be inhibited by normal human serum (Mollnes et al. Scand. J. Immunol. 20:157, 1984).
The present invention discloses a method which does not have the limitations or disadvantages of the prior art assays. Definitions of certain terms used herein may be found in Podack et al, Mol. Immunol. 21:589-603, 1984 which is incorporated herein by reference.